After fusion with the cellular plasma membrane or endosomal membranes, viral particles are generally too large to diffuse freely within the crowded cytoplasm environment. Thus, they will never reach the cell nucleus or the perinuclear areas where replication or reverse transcription usually takes place. It has been proposed that many unrelated viruses are transported along microtubules in a retrograde manner using the cellular dynein machinery or, at least, some dynein components. A putative employment of the dynein motor in a dynein‐mediated transport has been suggested from experiments in which viral capsid proteins were used as bait in yeast two‐hybrid screens using libraries composed of cellular proteins and dynein‐associated chains were retrieved as virus‐interacting proteins. In most cases DYNLL1, DYNLT1 or DYNLRB1 were identified as the dynein chains that interact with viral proteins. The importance of these dynein–virus interactions has been supported, in principle, by the observation that in some cases the dynein‐interacting motifs of viral proteins altered by site‐directed mutagenesis result in non‐infective virions. Furthermore, overexpression of p50 dynamitin, which blocks the dynein–dynactin interaction, or incubation of infected cells with peptides that compete with viral polypeptides for dynein binding have been shown to alter the viral retrograde transport. Still, it remains to be proved that dynein light chains can bind simultaneously to incoming virions and to the dynein motor for retrograde transport to take place. In this review, we will analyse the association of viral proteins with dynein polypeptides and its implications for viral infection.

Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P ؊2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P ؊2 position plus an acidic residue at the P ؊3 position (class II) can also occur, although interactions involving residues extending up to the P ؊7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P ؊3 and a Thr residue at P ؊2 . By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier. Neuronal NOS (nNOS)2 is expressed constitutively in specific neurons of the brain and in the spinal cord, peripheral nitrergic nerves, epithelial cells of various organs, pancreatic islet cells, and vascular smooth muscle (1). nNOS differs from the two other mammalian isoforms, endothelial NOS and inducible NOS by an additional ϳ300-residue N-terminal extension mostly involved in specific subcellular targeting. The N terminus of nNOS contains a PDZ domain (first found in the proteins PSD-95, Dlg, and ZO-1) (2), a ␤-hairpin module that associates to ␣1-syntrophin and PSD-95 (3), a DYNLL1 binding site (4), and a stretch known to bind to repeats R16/R17 of dystrophin (5). Brain nNOS is found in particulate and soluble forms in cells, and the differential subcellular localization of nNOS in various tissues may contribute to its diverse functions. Reinforcement of the idea that the subcellular targeting is exquisitely governed by this N-terminal extension came from the observation that an N-terminal deletion mutant of nNOS is an active, mislocalized enzyme (6). In the nervous system, the PDZ domain of PSD-95 is known to couple NMDA receptors to nNOS so that ⅐ NO release becomes reversibly regulated by Ca 2ϩ /calmodulin binding (1). PDZ domains are modula...

It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule–cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a β strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule‐associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.

It has been suggested that DYNLT1, a dynein light chain known to bind to various cellular and viral proteins, can function both as a molecular clamp and as a microtubule-cargo adapter. Recent data have shown that the DYNLT1 homodimer binds to two dynein intermediate chains to subsequently link cargo proteins such as the guanine nucleotide exchange factor Lfc or the small GTPases RagA and Rab3D. Although over 20 DYNLT1-interacting proteins have been reported, the exact sequence requirements that enable their association to the canonical binding groove or to the secondary site within the DYNLT1 surface are unknown. We describe herein the sequence recognition properties of the hydrophobic groove of DYNLT1 known to accommodate dynein intermediate chain. Using a pepscan approach, we have substituted each amino acid within the interacting peptide for all 20 natural amino acids and identified novel binding sequences. Our data led us to propose activin receptor IIB as a novel DYNLT1 ligand and suggest that DYNLT1 functions as a molecular dimerization engine bringing together two receptor monomers in the cytoplasmic side of the membrane. In addition, we provide evidence regarding a dual binding mode adopted by certain interacting partners such as Lfc or the parathyroid hormone receptor. Finally, we have used NMR spectroscopy to obtain the solution structure of human DYNLT1 forming a complex with dynein intermediate chain of ∼74 kDa; it is the first mammalian structure available.

Label-free differential scanning fluorimetry (DSF) is a relatively new method for evaluating the stability of proteins. It can be used as a screening tool for downstream applications such as crystallization. The method is attractive in that it requires miniscule quantities of proteins, it can be performed using intrinsic tryptophan and tyrosine fluorescence, and, with the right equipment, it is easy to perform. To date, the method has been used with proteins in liquid solutions and dispersions. It was of interest to determine if DSF could be used with membrane proteins in the lipid cubic phase (LCP), which increasingly is being used for crystallization in support of structure-function studies. The cubic phase is viscous. Furthermore, in coexistence with excess aqueous solution, as happens during crystallization trials, it can become turbid and scatter light. The concern was that these features may render the mesophase unsuitable for DSF analysis. However, using lysozyme and four integral membrane proteins we demonstrate that the method works with all tested proteins in solution and in the LCP. Of note is the observation that some of the test membrane proteins are more stable while others are less so in the mesophase. The method also works in ligand binding measurements. Thus, DSF should prove useful as an analytical tool for identifying host and additive lipids, detergents, precipitants and chemical probes that support the generation of quality crystals by the cubic phase method. Microscale thermophoresis was used to supplement the DSF study and was also shown to work with proteins in the mesophase. Measurements with lysozyme highlight the utility of the cubic mesophase as a model system in which to perform confinement studies.